E L S E V I E R Journal of Biotechnology 33 (1994) 135-146
journal of blotechnology
High level expression of the B. microplus Bm86 antigen in the yeast Pichia pastoris forming highly immunogenic
particles for cattle
Manuel Rodriguez a, Roger Rubiera a Manuel Penichet a, Raquel Montesinos a Jos6 Cremata a, Viviana Falcdn a, Giselle Sfinchez a, Ricardo Bringas a, Carlos Cordov6s b, Mario Vald6s b, Ricardo Lleonart a, Luis Herrera a,
Jos6 de la Fuente a,, a Mammalian Cell Genetics Division, Centro de Ingenieria Gen~tica y Biotecnolog[a, P. 0. Box 6162, Havana, Cuba;
b Estacidn Experimental de Parasitologfa, Havana, Cuba
(Received 14 April 1993; revision accepted 21 August 1993)
Abstract
Recently, a gene coding for the Bm86 tick gut glycoprotein was cloned, expressed in Escherichia coli and shown to induce an immunological response in cattle to damage ticks engorging on these animals (Rand et al., 1989). We report here the increased expression of the Bm86 antigen from the cattle tick Boophilus microplus in the methylotrophic yeast Pichia pastoris. The recombinant protein was obtained with a purity higher than 95% by a procedure with a high yield. The conducted biochemical studies demonstrated the antigen to be glycosylated and found to form particles of around 17 to 45 nm in diameter with enhanced immunogenic properties. Ticks engorging on vaccinated cattle were significantly damaged as a result of the immune response against the recombinant antigen. This system permits the obtainment in a high yield of the tick Bm86 antigen, in a glycosylated and particulated form.
Key words: Boophilus microplus; Tick; Antigen; P. pastoris; Gene expression
I. Introduction
The ectoparasite Boophilus microplus is a ma- jor veterinary problem affecting cattle health, as a debilitating agent itself and as a vector of severe diseases. The traditional control methods include the use ,of chemicals with partially successful re- sults, but this approach has certain drawbacks
* Corresponding author.
such as environmental and residue problems, the high incidence of acaricide resistance within tick populations on the field, and its high cost (Wil- ladsen and Kemp, 1988; Wikel, 1988). Cattle ac- quire a partial immunity to the ectoparasite after extensive natural exposure, due largely to an im- mediate hypersensitivity reaction to the tick which is, nevertheless, unable to prevent serious losses in cattle production. There have been different approaches to develop a method for tick control by the artificial induction of host-resistance, using
136 M. Rodffguez et al. /Journal of Biotechnology 33 (1994) 135-146
extracts from whole ticks, salivary glands and antigens associated with the saliva, so far unsuc- cessful (Wikel, 1981, 1988; Wikel and Alien, 1976; Geraint, 1991; Allen, 1979). The vaccination with 'concealed' gut antigens for tick control to pro- voke efficient tick gut damage was recently achieved using the B. microplus Bm86 glycopro- tein (Willadsen et al., 1989).
Employing the Bm86 antigen purified from the plasmatic membrane of tick gut epithelial cells, an induction of resistance to the ixodid infesta- tion was obtained by immunization. As a result, the blood digestion in these ticks was strongly and rapidly inhibited and the antibodies, together with other components of the immune system such as the complement, caused lysis of the tick gut epithelia (Willadsen et al., 1989; Johnston et al., 1986; Opdebeeck et al., 1988; Kemp et al., 1989; Willadsen et al., 1988). Partial protection against tick infestation was also obtained in cattle vaccinated with the recombinant Bm86 antigen derived from either Escherichia coli (Rand et al., 1989) or Aspergillus nidulans or niger (Turnbull et al., 1990) transformed cells, but the expression levels or the antigen recovery were apparently very low.
The methylotrophic yeast Pichia pastoris has been shown to be a suitable host for the indus- trial production of heterologous glycoproteins (Tschopp et al., 1987; Digan et al., 1989; Clase et al., 1991; Margolles et al., 1992). In this paper we report a high level expression of the B. microplus Bm86 antigen in P. pastoris. The described pro- cess is simple, easy to scale up and cost effective for industrial production, and allows the obtain- ment of a glycosylated and particulated antigen preparation. Ticks engorging on vaccinated cattle were significantly damaged as a result of the immune response against this recombinant anti- gen.
2. Materials and methods
2.1. Cloning of the Bm86 gene and construction of the yeast expression vector pPBm
The cloning strategy is depicted in Fig. 1. The 912 bp and 941 bp fragments corresponding to
the nucleotides 90-1001 and 975-1916 of the published sequence for Bm86 cDNA (Rand et al., 1989), respectively, were amplified using the primers 5 '-GTCTAGAGGAATCATTFGCTCT- GACT-FC (brat-l; upstream primer including an XbaI site), 5'-CCAGATCTrTAAGCACTTGAC- TTTCCAGGATC (bmt-2; downstream primer in- cluding a BgllI site) and 5'-GCTGCTATI'GTC- CATGGAAATCAAGG and 5'-CCTI'GATTTC- CATGGACAATAGCAGC (bmt-4 and bmt-3; internal primers including a unique NcoI site).
Boophilus microplus RNA was isolated from neoginas by the procedure of Chomczynski (Chomczynski and Sacchi, 1987). The poly A ÷ mRNA was purified by oligo (dT)-cellulose (Chirgwin et al., 1979) and diluted in RNase free water. The cDNA reaction was done employing 4 /xg of poly A + RNA in 100 /zl of a reaction buffer containing 50 mM Tris-HC1, 50 mM KC1, 6 mM MgCI2, 1 mM dXTPs, 50 U of RNAsin, 4/zg oligo dT primers, 10 mM DTT, pH 8.8 and 100 U of avian myeloblastosis virus reverse transcriptase (Promega, USA). After incubation at 42°C for 50 min, the reaction was stopped by freezing to -20°C. It was then thawed and 10/zl were mixed in a total volume of 100 /zl containing 10 mM Tris-HCl, 50 mM KC1, 2 mM MgC12, pH 8.3, 200 mM dXTPs, 0.1 nM of each oligonucleotide primer (bmt-1 and bmt-3 in the first reaction tube and bmt-2 and bmt-4 in the second reaction tube), and 3 U of Thermus aquaticus DNA polymerase (Heber Biotec, Cuba). Polymerase chain reaction (PCR) was conducted in 30 cycles of 20 s at 94°C (1 min for the first cycle), of 20 s at 55°C, and of 1 min at 72°C (3 min for the last cycle).
After phenol/chloroform extraction and etha- nol precipitation, the amplified products were subjected to agarose gel electrophoresis and ex- tracted from the gel (Maniatis et al., 1989). The 912 bp (Bm 5') and 941 bp (Bm 3') fragments (Fig. 1) were resuspended in TE (10 mM Tris-HC1, 1 mM EDTA, pH 8.1), containing 200/zM ATP and 2 U of T4 polynucleotide kinase (PNK, Heber Biotec, Cuba). The phosphorylation reaction was incubated for 1 h at 37°C and after phenol/ chloroform extraction and ethanol precipitation, phosphorylated fragments were cloned into a Bluescript plasmid digested with EcoRV to gen-
M. Rodffguez et al. /Journal of Biotechnology 33 (1994) 135-146 137
erate the plasmids pSBm5' and pSBm3' where both inserts were sequenced by the dideoxi chain termination method (Sanger et al., 1977).
Plasmids pSBm5' and pSBm3' were then di- gested with XbaI-NcoI and NcoI-BgllI, respec- tively, and the obtained fragments were inserted into an XbaI-BamHI pTrp digested vector (Es- trada et al., 1992) to assemble the Bm86 cDNA lacking the 3' terminal region in the resulting plasmid pTBm.
Employing the plasmid pTBm, a second PCR reaction was performed as previously described, but using the oligonucleotides 5'-GAATCATC- CATVI'GCTCT (bmt-5) and bmt-2 as 5' and 3' amplification primers, respectively, and 50 ng of plasmid DNA. The 1.83 kb amplified fragment was then extracted and phosphorylated as previ- ously described and inserted into the S1- nuclease-blunted NcoI site of the vector pPS 7 for expression of the Bm86 cDNA in the yeast P. pastor&. The plasmid pPS 7 is an integrative vector for the expression of heterologous proteins in P. pa~toris, which contains the 1.15 kb frag- ment from the methanol regulated alcohol oxi- dase (AOX1) promoter, followed by the signal peptide of the sucrose invertase (spSUC2) gene from Saccharomyces cerevisiae (Kaiser et al., 1987), the 960 bp fragment of the glyceraldehyde 3-phosphate dehydrogenase transcription termi- nator (GAPt) from S. cerevisiae, besides the histi- dine 3 (HIS 3) gene from S. cerevisiae as a selection marker in histidine requiring auxo- trophs P. pastoris strains. Furthermore, this plas- mid has a 3' sequence of 2.1 kb of the AOX1 gene, inserted in the pUC18 vector (Fig. 1). The resulting plasmid was designed as pPBm (Fig. 1).
2.2. Expression of the recombinant Bm86 (rBm86) antigen in yeast
The expression plasmid (pPBm) was EcoRI- SphI digested and the expression cassette trans- formed into histidine-requiring auxotrophs (HIS 3 - ), MP 36 P. pastoris strain (Yong et al., 1992) which acquired a His + phenotype following transformation. The transformants were analyzed by Southern blot to check for correct integration by replacement of the P. pastor& host AOX1
gene by the expression cassette of the pPBm to assure stable expression of a single copy inte- grant.
P. pastoris cells expressing Bm86 were grown in shake flasks with 50 ml of YPG medium (10 g yeast extract, 20 g peptone, and 2% w/v of glycerol for 1 1) at 30°C until obtaining an OD600n m
of 5-10. Then they were harvested, washed once with YP (glycerol free YPG medium), resus- pended in YP at approx. OD60on m 5 and incu- bated at 30°C for 3-4 d with methanol to induce expression. The culture media from methanol induced transformants were tested to assess the percentage of Bm86 expression by densitometric scanning of the Coomassie brilliant blue stained protein bands fractioned by SDS-PAGE using a LKB laser densitometer and also determined by ELISA. The production of Bm86 was examined in two batch-type runs, each one performed in a 5 1 Marubishi reactor equipped with monitor and controls for pH, dissolved oxygen, agitation speed, temperature and air flow measurements. Tem- perature was maintained at 30°C. Cell yields were estimated by the wet cell pellet weight. Inocula for the bioreactor runs were grown in a 1 1 Erlenmeyer flask containing 500 ml of YPG medium. Bioreactor cultures grown in the batch mode were propagated in YPG medium until the available glycerol was exhausted, then methanol was added to the culture for 96 h at a rate of 2.5 ml 1-1 h-1 for batch mode bioprocess.
Yeast cells were washed once in disruption buffer (50 mM sodium phosphate, 5 mM EDTA, 10% sucrose, 0.3 M NaCI, 1 mM 2-mercapto- ethanol, pH 7) and resuspended in the same buffer at a concentration of 350 g 1-1. The cells were disrupted for 14 min on a bead mill (Dy- nomill) equipped with a cold water cooling jacket at a flow rate of 20 ml min-1. The clarification of 1 1 of sample was conducted by centrifugation at 9000 × g for 20 min in RP 12-2 (HITACHI) ro- tor. The pellet was washed with 1 1 of washing buffer (50 mM sodium phosphate, 5 mM EDTA, 0.5% Triton X-100, 0.3 M NaC1, 1 mM 2- mercaptoethanol, pH 7), centrifuged at 9000 x g for 20 min and subjected to another three wash- ings with a buffer containing 50 mM sodium phosphate, 5 mM EDTA, 0.3 mM NaCI, 1 mM
138 M. Rodrfguez et al. / Journal o f Biotechnology 33 (1994) 135-146
Xbal
,/
B. microplus mRNA
cDNA
PCR
Nco I I bmt-4
Nc o I
] x ~ . . . . . ~ Bluescript ~cot I E c o R V
PNK 1 ÷ / ATP
p S B m 5' pTRP
NcoXball ~ Xbal ~BamHI
p T B m
PCR
Illllllll I
I Nco I BmM-y I
~St Z I
p S B m 3'
Ncol B.gttt
Nco I
Bin86 gene
GAPt
Bgl I1 His 3 , ~amHI Xba 1
3' AOXI
spsuc2
AOXI
p P B m 10.7 kb
His 3
AOX1
~ Nco I $1 nuc~
M. RodKguez et al. /Journal of Biotechnology 33 (1994) 135-146 139
2-mercaptoethanol, pH 7. The pellet resulting from the last centrifugation at 9000 × g for 20 min at room temperature was extracted by ho- mogenization with a Polytron (IKA T-50) in 2 1 of extraction buffer (8 M urea, 50 mM sodium phos- phate, 5 mM EDTA, 1 mM 2-mercaptoethanol, pH 7). All washings and subsequent steps were performed at room temperature. Combined cen- trifugation supernatants of the extraction were pumped into a Sephadex G-25 column previously equilibrated with a 50 mM sodium phosphate, 5 mM EDTA, pH 7 buffer. The total protein peak eluted from the column was sedimented by differ- ential precipitation in 35% NH4SO 4 to eliminate the excess of contaminants and the antigen was concentrated at 65% of NH4SO 4. The concen- trated antigen pellet was resuspended in a 50 mM sodium phosphate, pH 7 buffer for desalting in a Sephadex G-25 column equilibrated with 50 mM sodium phosphate, pH 7.
2.3. Preparation of a rabbit antisera against Bm86 peptides
For tracing the expression of the rBm86, the reported amino acid sequence of the natural pro- tein (Rand et al., 1989) was analyzed in the Hoops and Woods (1981) computer program, to select the possible antigenic determinants. Five immunogenic peptides were designed and synthe- sized by the method of simultaneous multiple peptide synthesis (Hughten, 1985). They were cleaved with liquid hydrogen fluoride, their com- position controlled by amino acid analysis and their purity assayed by a RP-C 18 4.6 × 100 mm HPLC column (J.T. Baker) at 34°C. The synthe- sized peptides had the following sequences: (1) E M T R G R L R R S V C K A G V S C N E N E Q S E C A K G Q(amino acids 474 to 505) (2) T T T K A K D K D P D P G K S S A(amino acids 612 to 628) (3) L S K H V L R K L Q A C E H ( a m i n o a c i d s 397 to 410)
(4) S S I C S D F G N E F C R N A (amino acids 21 to 35) (5) C D C G E W G A M N M T T R ( a m i n o acids 132 to 145)
Peptides were coupled to bovine serum albu- min (BSA) as carrier protein via the terminal cystein residue, using m-maleimidobenzoyl-N-hy- droxysuccinimide ester (Liu et al., 1979), and were used to immunize New Zealand white rab- bits. After obtaining preimmune sera, groups of two rabbits were immunized as follows: on day 0, each rabbit was injected intradermally (i.d.) with 200/zg of the peptide-albumin complex (PAC) in 1 ml of complete Freund's adjuvant (CFA); on day 14, they were injected i.d. with the same dose of the PAC emulsified with incomplete Freund's adjuvant (IFA) and on day 21, the animals were injected intraperitoneally with 200/zg of PAC in 1 ml of IFA. Rabbits were bled 15 d after the last immunization, serum was obtained, divided into aliquots, and stored at -20°C for later use.
The presence and specificity of anti-peptide antibodies were tested by dot-blot. Briefly, in different pieces of Hybond nitrocellulose sheets (Amersham) 5, 2.5, 0.62, 0.31, 0.15 and 0.08/zg of the PAC were spotted and 1 /~g of BSA and 20 /.~g of an unrelated coupled peptide were used as negative controls. Unbound reactive sites were blocked by incubation with 2% skim milk in PBS. The sheets of nitrocellulose were incubated with various rabbit sera diluted in 2% skim milk in PBS and the unbound material was removed by washing three times with 0.2% Tween 20 in PBS. Peroxidase-labeled affinity-purified goat anti-rab- bit IgG was added and the Hybond sheets were washed five times before substrate was added. Finally, the anti-peptides sera were pooled and used in Western blot analysis of cells extract from methanol induced MB 9 transformant.
2.4. Biochemical characterization of the rBm86 antigen
The molecular weight of rBm86 was estimated by polyacrylamide gel electrophoresis in the pres-
Fig. 1. Handling of mRNA from B. microplus to obtain the Bm86 gene and cloning into the P. pastoris expression vector pPS7 to construct the pPBm vector which directed the synthesis of rBm86. Details are described in Materials and Methods.
140 M. Rodffguez et al. /Journal of Biotechnology 33 (1994) 135-146
ence of sodium dodecyl sulfate (SDS-PAGE) and by molecular exclusion high performance liquid chromatography (HPLC) with a TSK-G 5000 PW (TOSO-HAAS Corp.) column in PBS with 0.25 M NaCI, as compared to 2400 kDa HBsAg particles of the hepatitis B virus, and with a TSK-3000 (TOSO-HAAS Corp.) column in denaturalizing conditions (50 mM Tris-HC1, 8 mM urea, 150 mM 2-mercaptoethanol, pH 7.2). The protein in 50 mM Tris-HC1 was centrifuged and mixed with 2% of phosphotungstic acid, to check it by elec- tron microscopy in the JEOL-JEM 2000 EX mi- croscope. For Western blot analysis, the protein was fractionated by SDS-PAGE at room temper- ature (Maniatis et al., 1989). Blots were devel- oped with an antipeptide antibody (1 : 5 dilution) and with a goat anti-rabbit IgG-colloidal gold complex. The N-terminal sequence analysis was performed on a KNAUER protein sequencer. The amino acid sequence was corroborated by mass spectrometry. The peroxidase-lectin staining of glycoproteins on membranes (Hsi et al., 1991; Garcla et al., 1992) was used to determine the glycosylation of rBm86. Briefly, 100 /zl of the sample were spotted on the membrane and the unbound reactive sites were blocked with 5% skim milk in TBS (50 mM Tris-HC1, 150 mM NaCI, pH 8.0), 1 h at 37°C. The membrane was then incubated for 1 h with concanavalin A-per- oxidase complex, 1:1000 diluted in PBS, and washed twice with TBS and once in TBS with 0.05% Tween 20. The development was per- formed with a 4-chloronaphtol solution (2 mg in 600/xl of methanol, 10 ml TBS and 10 jxl H202). The purified rBm86 was carboximethylated and the sulphydryl bonds reduced (Crestfield et al., 1963) before deglycosylation. For this reaction, 1 mg of rBm86 in 0.02% SDS was heated 10 min at 100°C, 10 mU of EndoH were added and incu- bated during 16 h at 37°C in 0.1 mM citrate buffer.
2.5. Cattle vaccination and challenge trials
Cattle were primarily grade Holstein, about 12 months old. They were purchased from cattle tick-free areas. Three groups of five heads were immunized with 100 /~g and 400 ~g of rBm86
antigen, and the third group was inoculated with 50 mM sodium phosphate, pH 7. All vaccination trials were done by intramuscular route, in Fre- und's complete adjuvant the first time and the second and third shots were at the 4th and 7th weeks in Freund's incomplete adjuvant. The serum antibodies to rBm86 were monitored by immunoassay (ELISA) and the antigens were ap- plied to the microtitre plate wells in a concentra- tion of 5/xg m1-1. The serum from each animal was tested for anti-Bm86 antibodies prior to vac- cination and several times later, by serial dilution of sera in the microtitre plate. After washing the plates, the antibody titres were determined with rabbit anti-bovine IgG immunoglobulins conju- gated with peroxidase. Cattle were infested 21 d after the last shot with 1000 B. microplus larvae applied individually to each animal per day dur- ing 3 d. After approx. 24 d, engorged adult fe- male ticks dropped from the cattle and each day ticks were collected, counted and weighed to calculate the mean weight per tick. The ticks were examined for obvious signs of damage, to determine the number of ticks that were visibly damaged on each day. All the female ticks that dropped from each animal were weighed, and assessed for egg laying capacity.
3. Results and discussion
3.1. Expression of the Brn86 gene in the yeast Pichia pastoris
The Bm86 gene fragment was obtained from B. microplus neogina by cDNA-PCR, using the bmt-1, brat-3 primers in the first reaction and bmt-2, brat-4 primers in the second. The two fragments Bm3' (912 kb) and Bm5' (941 kb) from the cDNA-PCR reactions, were cloned into the Bluescript vector to obtain the plasmids pSBm3' and pSBm5'. These plasmids were digested with XbaI-NcoI and NcoI-BgllI, respectively, to as- semble the Bm86 gene into the XbaI-BamHI site of the pTrp vector (Estrada et al., 1992), originat- ing the pTBm plasmid. Another PCR reaction was carried out with the pTBm as template, using bmt-5 and bmt-2 primers, to obtain a DNA frag-
M. Rodr(guez et aL /Journal of Biotechnology 33 (1994) 135-146 141
ment of 1.83 kb corresponding to the Bm86 gene, The Bin86 gene resulting from this PCR and which was cloned into the pPS7 vector to obtain sequenced by us, had three nucleotide changes at the pPBm (Fig. 1). DNA level, in the positions 239 (C to T), 364 (C
A MB9 MP36
t-bp
~3 °4 ~.5
.3
,3
B
I 5'AOX1 (1.1 kb) ~i
5'AOX1 (2.25 kb) AOX1 gene (1 kb)
P.pasto~
3'AOX1 (2kb) I
3'AOX1 (2.71 kb) 1
AOX1 gene (6 kb Eco RI fragment)
[ 5'AOX1 ~pSUC21Bm86gene ~ - ~ His3 ~ i YAOX1 I
Recombinant gene in the MB 9 transformant (8.9 kb Eco RI fragment) Fig. 2. (A) Southern blot analysis of AOX1 loci in MB 9 and MP 36 P. pastoris strains. The filter was hybridized with a 32pqabeled AOX1 promoter probe from the plasmid pPS 7(EcoRI-HindIII). The lanes contain 10/~g of EcoRI digested DNA from the MB 9 and MP 36 strains, size markers correspond to HindIII digested ADNA. (B) is a diagram of the double crossover leading to the replacement of the (6 kb) AOXlgene with the 7.72 kb EcoRI-SphI fragment containing the Bin86 expression cassette derived from pPBm.
142 M. Roddguez et al. /Journal of Biotechnology 33 (1994) 135-146
to T) and 377 (T to C), as compared to the reported sequence of the Bm86 gene (Rand et al., 1989), but only one of these changes in the position 364 implied a substitution of the amino acid Thr for Ile. This was confirmed by mass spectrometry analysis. This result could be a product of some heterogeneity of the tick popula- tion, although we can not discard the possibility of a misreading from the PCR reaction.
The P. pastoris expression vector pPBm con- tains the 608 amino acid coding sequence of the Bm86 gene, but lacking 23 amino acids of the carboxyterminal transmembrane region and the 19 amino acids fragment of the signal peptide sequence of the natural Bm86 glycoprotein. The expression cassette (pPBm) had 7.8 kb after SphI-EcoRI cleavage with free ends homologous to the AOX1 promoter and 3' sequences of the AOX1 gene from P. pastoris, to help in directing the integration of this expression cassette into the AOX1 locus of the P. pastoris genome, by homo- logous recombination resulting in the substitution of the endogenous AOX1 structural gene. South- ern blot hybridization analysis of the DNA iso- lated from the MB9 transformant showed that the AOX1 structural gene was replaced by a single copy SphI-EcoRI fragment from pPBm expression vector (Fig. 2), showing a Mut s phe- notype (Cregg and Malden, 1987).
The MB 9 transformant was grown until OD600n m 250 in a 5 1 culture medium bioreactor. After exhausting the glycerol from the culture medium, the cells were induced with methanol for 96 h. The cells were collected by centrifuga- tion and the rBm86 antigen was purified by a simple washed pellet procedure from the MB 9 disrupted pellet, with a very high percentage of recovery (80%) and purity. The antigen obtained by this method with a purity higher than 95%, allowed the determination of a single N-terminal sequence, Glu-Ser-Ser-Ile-Cys-Ser-Asp-Phe-Gly- Asn-Glu-Phe-Cys-Arg-Asn-Ala-Glu-Cys-Glu-Val- Val, which was in agreement with the reported Bm86 sequence (Rand et al., 1989). This also indicated the appropiate cleavage of the sucrose invertase signal peptide, used for secretion of the antigen.
The level of rBm86 secreted in P. pastoris
kDa
SDS-PAGE I
M.W. tBm86 MB9 MP36
Western-blot ]1 I
MP36 MB9
94
67
48
30
a0
Fig. 3. Expression analysis of rBm86. Coomassie blue-stained SDS-10.5% polyacrylamide gel (SDS-PAGE) showing total cell extracts from the MB 9 transformant and MP36 P. pastoris strains induced with methanol, in each lane were loaded 200 /zg of total protein. In lane 1, 5 /zg of purified rBm86 were loaded. Western blot analysis of cell extracts from methanol induced MB 9 and MP36 strains. For develop- ment, a rabbit anti-Bm 86 peptides polyclonal antiserum was used. Molecular weight markers (M.W.) correspond to phos- phorylase B (94 kDa), albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20.1 kDa) and fl-lactoalbumin (18 kDa).
during continuous biosynthesis on methanol was determined to be about 1.5 g l-1 of bioreactor medium at a cell density of 33 g l-1 (dry weight). This expression level was higher than those ob- tained in A. nidulans or A. niger (Tumbull et al., 1990). This high expression level in P. pastoris has also been reported for other recombinant proteins such as invertase (Tschopp et al., 1987), lysozyme (Digan et al., 1989), tetanus toxin (Clase et al., 1991) and a-amylase (Margolles et al., 1992).
3.2. Characterization of the rBm86 antigen
The rBm86 antigen produced in P. pastoris was analyzed by SDS-PAGE. This protein ap- peared in SDS-PAGE as a wide band, similar to a heterogeneous molecular species with size from 90 to 100 kDa (Fig. 3), but treatment with EndoH resulted in a single band of 70 kDa, correspond- ing to the molecular weight of the rBm86 protein
M. Rodrfguez et al. /Journal of Biotechnology 33 (1994) 135-146 143
without the presence of the N-linked oligosaccha- rides.
Three peptides numbered 3, 4 and 5 in Materi- als and Methods section were able to raise anti- bodies in rabbits. These anti-peptide antisera were pooled and used in the immuno-identifica- tion of the rBm86 protein by Western blot analy- sis. This assay showed that the 90-100 kDa band in the sample of medium from methanol induced cells was linked to rBm86 (Fig. 3).
The glycosylation of rBm86 was corroborated in a blotting assay with a concanavalin A-colloidal gold complex, because this complex has a very high affinity to the N-acetyl-D-glucosamine. This kind of N-linked oligosaccharides are the princi- pal type of glycosylation by P. pastoris yeast, in the same way that the high mannose oligosaccha- ride glycosylation is found in higher eukaryotic organisms (Ratner, 1989).
I
B
A Fig. 4. (A) Comparison between the HPLC exclusion patterns of recombinant HBsAg (peak I) and rBm86 particles (peak II). Running conditions: Column-TSK G 5000 PW, buffer-PBS and 0.25 M NaCl, 0.2 ml rain -1 flow rate. (B) Analysis of rBm86 particles by electron microscopy (× 40 000).
Fig. 5. Bovine serum antibody titres to rBm86 antigen deter- mined by ELISA in cattle vaccinated with 100 /xg (panel A) and 400 /zg (panel B). The panel (C) represents the weekly mean titres of the anti-Bin86 antibodies in both vaccinated and control groups.
144 M. Rodffguez et al. /Journal of Biotechnology 33 (1994) 135-146
In the TSK-G 5000 PW HPLC pattern without denaturalization, the retention time of rBm86 was 90 min (peak II, Fig. 4A), very similar to protein complexes such as the hepatitis B surface antigen (HBsAg), which was used as control. The latter has a retention time of 66 min (peak I, Fig. 4A) in the same running buffer as rBm86 (Fig. 4A). A sample of this protein was checked by electron microscopy to verify that the rBm86 anti- gen has a particulated structure, with particle size between 17-43 nm (Fig. 4B). Due to this reason the peak II did not have a symmetric shape like the peak I corresponding to HBsAg. This particle is larger than hepatitis B virus particles. How-
ever, in HPLC our protein ran slower than the hepatitis B surface antigen, perhaps because the rBm86 particle has a lower density than the HB- sag particle. Finally, the particulation and glyco- sylation characteristics are very important in sev- eral proteins because they could enhance the immunogenic performance of antigens in the im- munized animal.
3.3. Protection of cattle vaccinated with the rBm86 antigen
The particulated Bm86 antigen purified from P. pastoris was used in two different concentra-
Table 1 Results of the protection trial against B. rnicroplus in cattle immunized with different doses of rBm86 antigen
Animal Cumulative Cumulative Mean tick Damaged Cumulative Egg/tick tick number a tick weight b weight c ticks d egg weight e weight f
a Total number of ticks (all females) with approx. 0.8 cm of size were collected from each animal over the day 21 after the challenge. b Total weight of ticks collected from each animal. c (a) and (b) were used to calculate the mean weight per tick. d The percentage of tick damage is the sum of all ticks visibly damaged from the total number of ticks collected per animal. e The total weight of eggs produced by the surviving ticks under ideal conditions. All the female ticks collected from each animal, were incubated at 28°C and 80% of humidity, until eggs production had ceased. f The eggs weight was used to calculate the proportion of the tick weight that was converted into eggs.
M. Rodriguez et al. /Journal of Biotechnology 33 (1994) 135-146 145
tions to immunize five animals per group, which were subsequently chal lenged with ticks. In the group immunized with 400/xg rBm86, one animal did not comple te the exper iment and only four animals were challenged. The immunologic re- sponse was tested in each animal and the anti- body titres were followed in these experiments by ELISA. The animals immunized with 100/xg and 400 /zg had the same tick protect ion and im- munological response (Fig. 5). Engorged adult female ticks were collected on complet ion of the parasitic par t of the life cycle, approx. 23 d after the infection. Almost all ticks collected f rom both groups o f vaccinated animals changed their nor- mal gray color to a red color, as a signal of damage, due to the leakage of bovine erythro- cytes th rough the damaged tick gut wall into the hemolymph, and the average weight of ticks dropping after engorgement f rom vaccinated ani- mals was significantly reduced (50%). The ani- mals vaccinated with 100 ~ g doses presented a modest decrease in the number of ticks (31%), and a dramat ic effect of vaccinat ion was the reduct ion by 70% of the reproduct ive ability of ticks on vaccinated animals as compared to the control group (Table 1). The differences in the mean weight, percentage of damage and total egg product ion be tween both vaccinated groups and the control group, were statistically significant ( P < 0.01). These results were essentially similar to those repor ted using the rBm86 expressed in E. coli (Rand et al., 1989). We have shown that the MB 9 t ransformant of P. pastoris yeast con- stitutes a highly product ive source for obtaining large quantit ies of the rBm86 antigen, which have the right protective epi topes for damaging and decreasing B. microplus tick populations.
4. Acknowledgments
We thank Dr. Edua rdo Pen ton for critical reading of the manuscript . We wish to thank the following scientists f rom the staff of C I G B (Cent ro de Ingenier ia Gen&ica y Biotecnologla) for technical assistance in this project: J.J. Ma- drazo, V. Besada, V. More ra and V. Jim6nez, Camacho and Ivon f rom the Estaci6n Exper imen-
tal de Parasitologia, for main tenance o f tick cul- tures and moni tor ing the tick counts.
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